Tissue scaffold materials for tissue regeneration and methods of making

ABSTRACT

Disclosed herein are tissue scaffold materials with microspheres of one density embedded in hydrogel of a different density. The disclosed materials have improved ability to facilitate cellular invasion and vascularization for wound healing and tissue regeneration. The inventors have found that materials having components with different densities promotes invasion of cells, including desirable cells such as fibroblasts and endothelial precursor cells, into the scaffold.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 15/037,417, filed May 18, 2016, which is the 371National Phase of International Application No. PCT/US2014/066344, filedNov. 19, 2014, which claims priority from U.S. Provisional ApplicationNo. 61/906,131, filed Nov. 19, 2013, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

The optimization of cell guidance through autologous or artificialtissue scaffolds has long been a topic of great interest. The mostprevalent and thus far the most successfully applied off-the-shelf“tissue-engineered” products were all originally intended to serve asdermal replacement scaffolds. Commercially available scaffolds areacellular and thus share the common requirements of host cell invasionand vascularization to achieve durable incorporation. Because thisprocess is prolonged, requiring a minimum of several weeks forcompletion and necessitating obligatory dressing changes, woundimmobilization, and nursing care, there is significant interest indeveloping better scaffolds that could optimize the rate of cellularinvasion. (Eppley, Plast Reconstr Surg. 107:757-762 (2001); Wong et al.,Plast Reconstr Surg. 121:1144-1152 (2008)).

Currently available acellular dermal replacements can be categorizedinto two broad groups: products derived from decellularized dermis, andsynthetic products based on naturally-derived hydrogels (Truong et al.J. Burns Wounds 4:e4 (2005)).

Commercially available decellularized dermal products are made ofdecellularized cadaveric porcine or human dermis. As a result of thedecellularization process, these products contain an internal network ofmicrochannels with an intact basement membrane that are the remnants ofthe native dermal microvasculature.

INTEGRA (Integra LifeSciences, Plainsboro, N.J.), another commonlyapplied dermal regeneration template, is comprised of a synthetic“dermal” porous layer of cross-linked type I bovine collagen andchondroitin-6-sulfate covered by an “epidermal” semi-permeable siliconesheet. Following implantation, the silicone sheet is replaced withsplit-thickness autograft once the dermal layer has vascularized (Yannaset al., Science 215:174-176 (1982)). Unlike decellularized dermalproducts, INTEGRA is representative of products without an internalvascular structure and is instead characterized by its random porosity(mean pore diameter 30-120 μm) (van der Veen et al., Burns 36:305-321(2010)).

The use of currently available tissue replacement scaffolds is notwithout substantial associated cost. For example, the production ofdecellularized dermal products requires tissue acquisition andharvesting, as well as decellularization and sterilization processes (Nget al., Biomaterials 25:2807-2818 (2004)). In addition, commerciallyavailable tissue scaffolds are avascular and prone to high failure rateswhen used in complex settings, such as irradiated wounds or those withexposed hardware or bone. In such complex settings, neovascularizationis insufficient using existing tissue replacement products.

Improved tissue scaffolds that promote optimal cellular invasion andvascularization of new and surrounding tissue are highly desired in theart.

BRIEF SUMMARY OF THE DISCLOSURE

Disclosed herein is a type of tissue scaffold material made of ahydrogel with embedded microspheres. In the disclosed tissue scaffolds,the microspheres have a different or greater density (w/v) of polymerrelative to the density of the hydrogel, which differential densityfacilitates cellular invasion into the tissue scaffold. In oneembodiment, the hydrogel includes a first polymer and the microspheresinclude a second polymer, the microspheres are embedded in the hydrogel,and the microspheres have a greater density than the hydrogel.

The first and second polymers can be independently selected from thegroup consisting of of collagen, gelatin, elastin, hyaluronate,cellulose, fibrinogen, poly(lactic-co-glycolic acid) (PLGA),poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(caprolactone),poly(butylene succinate), poly(trimethylene carbonate),poly(p-dioxanone), and poly(butylene terephthalate); a polyester amide,a polyurethane, poly[(carboxyphenoxy) propane-sebacic acid],poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloyl chloride], a poly(ortho ester), apoly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester,poly((3-hydroxyalkanoate), and a tyrosine derived polycarbonate. Inexamples, the microspheres can contain 0.2% to 2.0%, 0.4% to 1.2%, 0.6%to 1.0%, or 1.0% w/v of the second polymer. In a particular example, thesecond polymer is collagen. The microspheres can be between 50-250 μm indiameter. The microspheres can fill at least about 50%, 60%, or 70% byvolume of the tissue scaffold material. The microspheres can alsocontain bioactive factors, but in some embodiments, the microspheres donot contain bioactive factors. The bioactive factors can promote one ormore of cellular invasion, cellular growth, or vascularization.

In one example, the hydrogel contains collagen. In some examples, thehydrogel contains collagen in an amount of 0.1% to 0.6%, 0.2 to 0.4%, or0.3% w/v. The tissue scaffold material can have microspheres with 0.2%to 2.0%, 0.4% to 1.2%, 0.6% to 1.0%, or 1.0% w/v collagen, embedded in ahydrogel with 0.1% to 0.6%, 0.2 to 0.4%, or 0.3%% collagen w/v. In oneembodiment, the tissue scaffold material has microspheres with 0.6-1.0%w/v collagen, embedded in a hydrogel containing 0.3% w/v collagen.

The tissue scaffold material of any of the above embodiments can be inthe form of a sheet or in a flowable form. The material can be, forexample, in the form of a sheet with a depth of 0.5-3.0 mm, or about1.0-2.0 mm. The disclosed tissue scaffold materials can be used in amethod of wound healing or tissue regeneration in a subject.

Further disclosed herein are methods to promote wound healing or tissueregeneration in a subject in need thereof, by applying the tissuescaffold material as disclosed above or herein to a wound or tissue ofthe subject. The tissue scaffold material can be applied, for example,to an area of the subject with exposed bone, hardware, or necrotictissue.

Also disclosed herein are methods of making a tissue scaffold material.The methods involve the steps of: (a) providing a first composition withmicrospheres, and a second composition with a polymer material, thefirst composition having a different density than the secondcomposition; (b) mixing the first and second compositions; and (c)causing crosslinking of the polymer material in said mixture, to form ahydrogel with embedded microspheres. The first and second compositionscan each contain collagen, such as human or bovine collagen, as apolymer. The collagen can be neutralized collagen. The microspheres cancontain 0.4% to 1.2%, or 0.6-1.0% w/v of collagen. The microspheres canfurther contain bioactive factors. The second composition can contain0.1% to 0.6%, or 0.3% w/v of collagen. Crosslinking can be accomplished,for example, by thermal methods.

Also disclosed are tissue scaffold materials produced by the methodsprovided above and further disclosed herein, and wound dressingscomprising such tissue scaffold materials.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C. At seven days post-implantation, cells infiltrate MSSscaffolds (C) but do not infiltrate 1% bulk alone (A) and poorlyinfiltrate 0.3% bulk (B).

FIGS. 2A-2C. At fourteen days post-implantation, cells show excellentinfiltration of MSS scaffolds (C) but do not infiltrate beyond outerportion of 1% bulk (A) and show only modest infiltration of 0.3% bulk(B).

FIGS. 3A-3D. At seven days post-implantation, cells show more completeinfiltration of MSS scaffolds with 1% microspheres in 0.3% bulk (C), and0.6% microspheres in 0.3% bulk (D), with less infiltration of 0.4%microspheres in 0.6% bulk (A) and 0.4% microspheres in 0.2% bulk (B).

FIGS. 4A-4B. At seven and fourteen days post-implantation, cellularinfiltration of 1% microspheres in 0.3% bulk (blue staining, DAPI)includes endothelial precursor CD31+ cells (red staining).

FIGS. 5A-5D. Seven days post-implantation. (A-C), identification of MSS,0.3% bulk, 1% bulk and INTEGRA scaffolds in mouse. (D), relative sizesof scaffolds after implantation.

FIGS. 6A-6C. Seven days post-implantation, cells infiltrate MSS scaffoldall the way to the center of the scaffold (A) but do not infiltrate 1%bulk except where scaffold is split (B) and poorly infiltrate 3% bulk(C).

FIG. 7. Seven days post-implantation. DAPI nuclear staining (blue)demonstrating cell invasion to the center of the MSS and CD31+endothelial precursors (red).

FIGS. 8A-8E. Fourteen days post-implantation. (A-D), identification ofMSS, 0.3% bulk, 1% bulk and INTEGRA scaffolds in mouse. (E), relativesizes of scaffolds after implantation.

FIGS. 9A-9D. Fourteen days post-implantation. (A), significant cellularinvasion in MSS scaffold. (B), 1% collagen with minimal invasion (exceptalong fissures). (C), 0.3% collagen scaffold with sparse invasion. (D),INTEGRA at 14 days also with less robust appearing invasion.

FIG. 10. Cell count per unit scaffold area shows that significantly morecells invaded the MSS scaffold at 7 and 14 days (approximately 7 cellsand 10 cells per area, respectively) relative to 1% hydrogel(approximately 3 and 5 cells per unit area) and 0.3% hydrogel(approximately 3 and 7 cells per unit area).

FIGS. 11A-11D. Twenty eight days post-implantation. (A-C),identification of MSS, 0.3% bulk, 1% bulk and INTEGRA scaffolds inmouse. (D), relative sizes of scaffolds after implantation. Note 0.3%hydrogel is significantly shrunken.

FIGS. 12A-12D. Twenty eight days post-implantation. (A), excellentcellular invasion in MSS scaffold. (B), 1% collagen maintains minimalinvasion (except along fissures). (C), 0.3% collagen scaffold showsuniform moderate invasion. (D), INTEGRA also shows reasonable invasion.

FIG. 13. Scanning electron microscopy of microspheres.

DETAILED DESCRIPTION OF THE DISCLOSURE

Disclosed herein are tissue scaffold materials with improved ability tofacilitate cellular invasion and vascularization for wound healing andtissue regeneration. The inventors have found that materials havingcomponents with different densities promotes invasion of cells,including desirable cells such as fibroblasts and endothelial precursorcells, into the material.

The terms “tissue scaffold”, “tissue scaffold material”, “dermalsubstitute”, “dermal substitute material” and “material” are usedinterchangeably herein to refer to a cell growth support structure madeof biocompatible polymer. These materials are capable of regeneratingdamaged tissues by providing a biocompatible template that promotescellular invasion and tissue regeneration.

The tissue scaffold materials disclosed herein are composed of ahydrogel support, which is filled with microspheres. The microsphereshave a density (the density being measured as weight by volume or w/v)that differs from the density of the hydrogel in which the microspheresare embedded. In a preferred embodiment, the microspheres have a greaterdensity than the hydrogel. However, the microspheres can have a lowerdensity than the hydrogel.

Throughout this application, the terms “about” and “approximately”indicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects. In one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

Polymers

The microspheres, hydrogels, and compositions disclosed herein containpolymers. The microspheres and hydrogels can contain the same polymer,or can contain different polymers from one another. A “polymer” is amacromolecule composed of repeating subunits. Suitable polymer materialsfor tissue engineering include natural polymers, such as collagen,gelatin, elastin, hyaluronate, and cellulose; fibrinogen; and syntheticpolymers, including polyesters such as poly(lactic-co-glycolic acid)(PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA),poly(caprolactone), poly(butylene succinate), poly(trimethylenecarbonate), poly(p-dioxanone), and poly(butylene terephthalate);polyester amides, such as HYBRANE S1200 (DSM, The Netherlands);polyurethanes, such as DEGRAPOL (Abmedica, Italy); polyanhydrides, suchas poly[(carboxyphenoxy) propane-sebacic acid]; polyphosphoesters, suchas poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloyl chloride]; poly(ortho esters);poly(alkyl cyanoacrylates); polyethers, such as poly(ethylene glycol);microbial polyesters, such as poly((3-hydroxyalkanoate); and poly(aminoacids), such as tyrosine derived polycarbonate (for review, see Marin etal., Int. J. Nanomed. 8:3071-3091 (2013)). In one embodiment, thepolymer is selected from the group consisting of collagen, hyaluronicacid, poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA),and poly(lactic acid) (PLA). Preferred polymers are collagen andcollagen-based biomaterials, including collagen types I, II, III, IV,and V. Particularly preferred for use in human subjects are human andbovine collagens, such as human or bovine type I collagen.

Microspheres

“Microspheres” are small particles, made of a polymer. The term“microspheres” as used herein encompasses small particles that can bespherical or non-spherical; accordingly, any reference to “microspheres”in this application can be used interchangeably with the term“microstructures”, as the microspheres disclosed herein include bothspherical and non-spherical small particles. Although microspheres canencompass any diameter from 1 μm-1 mm, microspheres as disclosed hereintypically have a diameter of between 10-500 μm in diameter, between50-250 μm in diameter, between 50-150 μm in diameter, or between 100-200μm in diameter, for example. In one embodiment, microspheres in a tissuescaffold material are fairly uniform in size and shape, for example, allthe microspheres in a given scaffold can be roughly spherical and have adiameter of about 50-150 μm, or about 100-200 μm in diameter. In anotherembodiment, microspheres in a given scaffold can differ in shape, forexample, some can be flattened, curved, oblong, or irregularly shaped,while others can be spherical. In another embodiment, microspheres in agiven scaffold can differ in size, for example, differing in size from10-500 μm, or even 1-1000 μm in diameter.

In some examples, the microspheres are made of 0.2% to 2.0%, 0.4% to1.2%, 0.4% to 0.8%, or 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,or 1.0% w/v of a polymer selected from collagen, gelatin, elastin,hyaluronate, cellulose, fibrinogen, poly(lactic-co-glycolic acid)(PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA),poly(caprolactone), poly(butylene succinate), poly(trimethylenecarbonate), poly(p-dioxanone), and poly(butylene terephthalate); apolyester amide, a polyurethane, poly[(carboxyphenoxy) propane-sebacicacid], poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloyl chloride], a poly(ortho ester), apoly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester,poly(β-hydroxyalkanoate), and a tyrosine derived polycarbonate. In aspecific embodiment, the polymer is collagen.

The microspheres can further include bioactive factors in addition tothe polymer. A “bioactive factor” can be a small organic molecule, anucleic acid, or a polypeptide that can stimulate or promote one or moreof cellular invasion, cellular growth, angiogenesis, vascularization,nerve regeneration, or cellular differentiation. The bioactive factorcan be, for example, a growth factor contained within the micro sphereor mixed with the polymer matrix of the microsphere prior to preparingthe tissue scaffold material. In one example, the bioactive factor is agrowth factor selected from the group consisting of nerve growth factor(NGF), vascular endothelial growth factor (VEGF), platelet derivedgrowth factor (PDGF), neurotrophin-3 (NT-3), brain derived growth factor(BDNF), acidic and basic fibroblast growth factor (FGF), pigmentepithelium-derived factor (PEDF), glial derived growth factor (GDNF),angiopoietin, and erythropoietin (EPO). In another example, thebioactive factor is a nucleic acid, such as antisense siRNA molecule. Inother embodiments, the microspheres do not include other bioactivefactors.

Hydrogels

The term “hydrogel” refers to a broad class of polymeric materials whichare swollen extensively in water, but which do not dissolve in water.Generally, hydrogels are formed by polymerizing a hydrophilic monomer inan aqueous solution under conditions where the polymer becomescrosslinked so that a three dimensional polymer network is formed whichis sufficient to gel the solution. Hydrogels are described in moredetail in Hoffman, D. S., “Polymers in Medicine and Surgery,” PlenumPress, New York, pp 33-44 (1974).

The hydrogels disclosed herein can be composed of the polymers providedabove. In examples, the hydrogel contains 0.1% to 0.6%, 0.2 to 0.4%, or0.3% w/v of a polymer selected from the group consisting of collagen,gelatin, elastin, hyaluronate, cellulose, fibrinogen,poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA),poly(lactic acid) (PLA), poly(caprolactone), poly(butylene succinate),poly(trimethylene carbonate), poly(p-dioxanone), and poly(butyleneterephthalate); a polyester amide, a polyurethane, poly[(carboxyphenoxy)propane-sebacic acid], poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloyl chloride], a poly(ortho ester), apoly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester,poly((3-hydroxyalkanoate), and a tyrosine derived polycarbonate. In oneexample, the hydrogel contains collagen. In some examples, the hydrogelcontains collagen in an amount of 0.1% to 0.6%, 0.2 to 0.4%, or 0.3%w/v.

Methods of Making Tissue Scaffold Materials

Also disclosed herein are methods of making a tissue scaffold material.The methods involve the steps of: (a) providing a first composition withmicrospheres, and a second composition with a polymer material, thefirst composition having a different density than the secondcomposition; (b) mixing the first and second compositions; and (c)causing crosslinking of the polymer material in said mixture, to form ahydrogel with embedded microspheres.

To make the scaffolds, suitable polymers are incorporated intocompositions for production. Suitable polymers include natural polymers,such as collagen, gelatin, elastin, hyaluronate, and cellulose;fibrinogen; and synthetic polymers, including polyesters such aspoly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA),poly(lactic acid) (PLA), poly(caprolactone), poly(butylene succinate),poly(trimethylene carbonate), poly(p-dioxanone), and poly(butyleneterephthalate); polyester amides, such as HYBRANE S1200 (DSM, TheNetherlands); polyurethanes, such as DEGRAPOL (Abmedica, Italy);polyanhydrides, such as poly[(carboxyphenoxy) propane-sebacic acid];polyphosphoesters, such as poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloyl chloride]; poly(ortho esters);poly(alkyl cyanoacrylates); polyethers, such as poly(ethylene glycol);microbial polyesters, such as poly((3-hydroxyalkanoate); and poly(aminoacids), such as tyrosine derived polycarbonate (for review, see Marin etal., Int. J. Nanomed. 8:3071-3091 (2013)). In one embodiment, thepolymer is selected from the group consisting of collagen, hyaluronicacid, poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA),and poly(lactic acid) (PLA). Preferred polymers are collagen andcollagen-based biomaterials, including collagen types I, II, III, IV,and V. Particularly preferred are human and bovine collagens. Bovinetype I collagen is commercially available, for example, from LifeTechnologies, Inc. Human type I collagen is available, for example, inlyophilized form or solution, as VITROCOL (Advanced Biomatrix, Inc., SanDiego, Calif.). Recombinant human collagen is available, for example, asCOLLAGE Collagen (CollPlant Ltd., Ness-Ziona, Israel).

Collagen can be derived from various sources, such as human or bovinetissue. Collagen can be autologous to the subject for whom the tissuescaffold is to be administered, and can be extracted, for example, fromthe skin of the subject. Once a suitable biological sample (such asskin, placenta, tendon, or cultured cells) is procured, collagen can beextracted from the sample by known techniques to form a stock solution.See, for example, Epstein, J. Biol. Chem. 249:3225-3231 (1974). Stocksolutions of collagen can include collagen in a suitable solution,containing, for example, 0.1% acetic acid, or Earle's or Hank's salts,L-glutamine, HEPES, and sodium bicarbonate. An example of a suitablemedium is a Medium 199 (M199)-based medium. Such media are commerciallyavailable, for example, from Sigma-Aldrich, Life Technologies, and othercell culture media vendors. Collagen is generally kept at a stockconcentration higher than the final concentration, such asconcentrations of 0.2%-1.6% collagen, preferably 0.3-0.5% collagen forthe hydrogel, and 0.6-2.0% collagen for the microspheres. Collagensuitable for use in the disclosed methods is also commerciallyavailable.

In some embodiments, collagen is neutralized before use. Collagen can beneutralized by mixing a stock solution of collagen with sodium hydroxideto reach a pH of 7.2-7.6, preferably pH 7.4. This mixture can beoverlayed with oil, such as mineral oil, preferably at least 5 volumesof oil per volume of collagen with NaOH, and stored with refrigerationuntil use.

To make microspheres, a polymer (e.g., collagen) composition with oiloverlay is mixed at high speed to form an oil-in water emulsion. Thepolymer composition can further contain at least one type of bioactivefactor as disclosed hereinabove. The emulsion is then subject torepeated washings with increasing concentrations of ethanol, forexample, a first wash with 50% ethanol, a second wash with 80% ethanol,and a third through fifth wash with 100% ethanol. The first washcomprises mixing (such as by stirring at 800-1500 rpm for 20-40 minutes)with at least 5 volumes of ethanol per volume of collagen solution,centrifuging the mixture at 2500-3500 rpm for 5-10 minutes, and removingthe oil and alcohol layers. Subsequent washes include mixing with atleast 5 volumes of ethanol per volume of collagen solution, centrifugingthe mixture at 2500-3500 rpm for 5-10 minutes, and removing the alcohollayer. After the alcohol washes, the collagen is then washed three tofive times with at least 5 volumes of cold saline, such as phosphatebuffered saline (PBS). After removal of the final saline wash, thecollagen microsphere composition formed by the washes is ready for use.

The polymer used for the hydrogel can be the same or different from thepolymer used to make the microspheres. In some embodiments, the polymerfor the hydrogel is the same as the polymer for the microspheres. Inother embodiments, the polymer for the hydrogel is different from thepolymer for the microspheres. In a preferred embodiment, the polymerused for both the microspheres and the hydrogel is collagen. However,whether the microspheres and hydrogel have the same or differentpolymer, the density of the polymer (w/v) in the microspheres willdiffer from the density of polymer (w/v) in the hydrogel “bulk”.

To make collagen hydrogel “bulk” scaffolds, a collagen stock solution ismixed with sodium hydroxide to reach a pH of 7.2-7.6, preferably pH 7.4.This collagen composition is then ready for use.

To make the tissue scaffold materials, the first composition, containingmicrospheres, is added to a mold or shaping platform. The secondcomposition that will form the hydrogel, containing a polymer material,is added to the first composition. The compositions are mixed, such asby stirring or pipetting, to achieve uniform mixing. The mixture is thencross-linked by standard methods suitable for crosslinking polymers,such as by thermal (incubating at 35-45° C., preferably 37° C., for20-40 minutes) or chemical methods. Following cross-linking, the tissuescaffold material can be used immediately or stored for future use.

Tissue Scaffolds and Dressings

Also disclosed are tissue scaffold materials produced by the methodsprovided herein. The microspheres and hydrogel making the tissuescaffold each contain a polymer selected from the group consisting ofcollagen, gelatin, elastin, hyaluronate, cellulose, fibrinogen,poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA),poly(lactic acid) (PLA), poly(caprolactone), poly(butylene succinate),poly(trimethylene carbonate), poly(p-dioxanone), and poly(butyleneterephthalate); a polyester amide, a polyurethane, poly[(carboxyphenoxy)propane-sebacic acid], poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloyl chloride], a poly(ortho ester), apoly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester,poly((3-hydroxyalkanoate), and a tyrosine derived polycarbonate. In oneembodiment, the microspheres and hydrogel of the disclosed tissuescaffold material each contain collagen, such as human or bovinecollagen, as a polymer. The collagen can be neutralized collagen. Thetissue scaffold material can be in a flowable form suitable forinjection into a subject, or in a sheet form, for example, a sheet witha depth of 0.5-3.0 mm, or 1-2 mm.

In particular examples, the tissue scaffold material can havemicrospheres with 0.2% to 2.0%, 0.4% to 1.2%, 0.6% to 1.0%, or 1.0% w/vcollagen, embedded in a hydrogel with 0.1% to 0.6%, 0.2 to 0.4%, or0.3%% collagen w/v. Microspheres have a density different from,typically great than, that of the hydrogel. The difference between thedensities should be at least 25%. In some embodiments, the difference isat least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more,when comparing the density of microspheres relative to the density ofcollagen. In one embodiment, the tissue scaffold material hasmicrospheres with 0.6-1.0% w/v collagen, embedded in a hydrogelcontaining 0.3% w/v collagen. In another embodiment, the microspheresfill at least about 50%, 60% or 70% of the volume of the tissue scaffoldmaterial. In a further embodiment, the microspheres contain bioactivefactors, such as growth factors.

Further disclosed are wound dressings and medical products into whichthe disclosed tissue scaffold material is integrated. The tissuescaffold material may be embedded into the dressing, or deposited on oneside of the dressing. The dressing can further include one or more ofsilicone, gauze, or other covering, and/or an antibiotic,anti-inflammatory or pain reducing agent or other ointment to facilitatehealing or reduce pain.

The tissue scaffold product can be further suitably packaged, such as insterile packaging, for use in wound healing or tissue regeneration.

Methods of Treatment

Further disclosed herein are methods to promote wound healing or tissueregeneration in a subject in need thereof, by applying the tissuescaffold material as disclosed herein to a wound or tissue of thesubject. The tissue scaffold material can be applied, for example, toany area of the subject in which tissue regeneration is desired, such asapplication to an open wound or during the course of a surgicalprocedure. In preferred embodiments, the disclosed tissue scaffolds areapplied to areas of the body with exposed bone, hardware, or necrotictissue.

The tissue scaffolds disclosed herein can be removed or remain in place.The polymer can be biodegradable and in such cases will graduallydissolve, leaving behind a new network of cells and vasculature formedfrom the subject's cells.

As used herein, the terms “subject” and “patient” are usedinterchangeably and refer to an animal, including mammals such asnon-primates (e.g., cows, pigs, horses, cats, dogs, rats etc.) andprimates (e.g., monkey and human).

The present disclosure is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1. Production of Microsphere/Hydrogel Scaffolds

Collagen type I was extracted from rat tail samples using standardtechniques. Skin was removed from rat tails using sharp dissection anddiscarded. Then, starting from the distal end of the tail, tendons wereextracted by breaking a joint within the vertebrae and pulling upward onthe distal vertebrae until the distal vertebrae with attached tendonseparated from the remaining proximal tail. The vertebrae was thensharply dissected from the tendon and discarded. Next, the tendon wasplaced in 70% ethanol. This was repeated until all joints within thetail were broken and tendons extracted. The extracted tendons werecollected, weighed and placed in a sterile 1 L container. Thereafter,0.1% acetic acid was added to the tendons to reach a final concentrationof 75 ml of acetic acid/g of tendon in order to arrive at a stockcollagen solution of 15 mg/mL (1.5% w/v) type I collagen. The collagenstock was then stored at 4 C and agitated for approximately 1 minutedaily for at least 72 hours.

After 72 hrs, the collagen stock was aliquoted into 50 mL conical tubes,centrifuged at 4° C. and 8800 rpm for 90 minutes, and any pellet removedand discarded. The final 15 mg/mL (1.5% w/v) collagen stock was thenplaced in a standard lyophilizer and lyophilized for at least 72 hours.Following lyophilization, collagen stock was stored at −4° C. until use.Upon use, this lyophilized collagen was resuspended in 0.1% acetic acidto a concentration of 10 mg/mL (1% w/v). This resuspended collagen wasagitated daily (for approximately 1 min) for 3 days prior to use. Stocksolutions of 1.5% (w/v) collagen and 0.384% (w/v) collagen were used tocreate microspheres and 0.3% hydrogels, respectively.

To neutralize collagen to make 1% microspheres, 2 ml of 1.5% collagenwas mixed with 656 μl of 1×M199 medium (Gibco/Life Technologies, Inc.),300 μl of 10×M199 medium, and 44 μl NaOH (or more NaOH as needed toadjust pH to 7.4), on ice. This mixture was overlayed with at least 5times volume (e.g., 15 ml) of mineral oil, and stored at 4° C. untiluse.

To produce microspheres, neutralized collagen with oil overlay was mixedby high-speed vortexing for about 5 minutes to create a water-in-oilemulsion. The emulsion was then poured into a flask, combined with atleast 5 volumes of 50% ethanol per volume of collagen solution minusoil, and stirred with a stir bar at 1100 rpm for 30 minutes. The stirredmixture was then poured into a 50 ml tube, and centrifuged at 3200 rpmat 4° C. for 7 minutes to form oil and ethanol layers with a thin layerof collagen between the oil and alcohol layers. The oil and alcohollayers were removed, the collagen layer was washed with 5 volumes of 80%ethanol, vortexed and centrifuged as above, alcohol layer removed,washed with 5 volumes of 100% ethanol, vortexed and centrifuged, and thealcohol layer removed. The collagen was then washed for three roundswith 5 volumes of cold PBS, vortexed and centrifuged, and PBS removed.During this process, collagen microspheres are formed.

To prepare collagen “bulk” for hydrogels, 391 μl of 0.384% collagen wasmixed with 50.8 μl of 1×M199 medium, 50 μl of 10×M199 medium, and 8.6 μlNaOH (or more NaOH as needed to adjust pH to 7.4), on ice. This mixturecan then be used to make scaffolds, as follows.

To make the scaffolds, molds were used with a diameter of 7 mm and adepth of 2.5 mm to create a scaffold of approximately 96 mm³. To makemicrosphere scaffolds, microspheres produced by the methods above werepipetted into each well to fill each well about half full. One drop ofthe collagen bulk was added to each well, and mixed with themicrospheres by stirring, to form a hydrogel embedded with microspheres.The scaffolds were then cured at 37° C. for 30 minutes. Phosphatebuffered saline (PBS) was overlayed on the cured scaffolds to preventfurther drying. To make “bulk” scaffolds, collagen bulk was added to themolds, without microspheres, to approximately the same level as thescaffolds with microspheres. The scaffolds were cured as above andoverlayed with PBS.

According to Kepler's conjecture of close-packed spheres, approximately74% of the volume of the scaffold should be comprised of higher densitymicrospheres, with the remaining volume taken up by the bulk collagenhydrogel.

Example 2. Microsphere Containing Scaffolds Promote CellularInfiltration

Scaffolds were produced one day prior to implantation. Scaffolds wereimplanted subcutaneously in the dorsa of 8 week old wild-type C57bl/6mice. 3 mice were implanted with 4 total scaffolds as follows: Two 1%microspheres in 0.3% bulk scaffolds; one 1% bulk scaffold as a control;one 0.3% bulk scaffold as a control. All mice were sacrificed andharvested for histological analysis after 7 or 14 days. Hematoxylin andeosin (H&E) staining was performed on tissue samples embedded in optimalcutting temperature compound (OCT) medium, to identify cellularinfiltration into scaffolds.

After 7 days of implantation, the microsphere scaffolds (MSS) showsubstantial and uniform cellular invasion spanning the entire depth ofthe scaffold (FIG. 1C). Comparatively, cells sporadically and onlypartially invaded the 0.3% control scaffolds (FIG. 1B), and failed toinvade the 1% control scaffolds, instead proliferating along theperiphery of the scaffolds (FIG. 1A).

After 14 days of implantation, MSS revealed robust cellular invasionspanning the scaffold depth (FIG. 2C). Comparatively, cells sporadicallyinvaded 0.3% (w/v) collagen scaffolds (FIG. 2B) and failed to invade 1%(w/v) collagen scaffolds altogether, instead remaining confined to theperiphery (FIG. 2A).

Example 3. Different Densities of Microspheres Relative to HydrogelDensity Promote Cellular Infiltration

Microsphere scaffolds with different densities (w/v) of collagen inmicrosphere (MS) and hydrogel (H) were prepared as follows: (A) 1%collagen MS in 0.3% H; (B) 0.6% MS/0.3% H; (C) 0.4% MS/0.2% H; (D) 0.4%MS/0.6% H. See, Table 1.

TABLE 1 Densities of Microsphere Scaffolds Microsphere Collagen Density(w/v) Bulk Collagen Density (w/v)   1% 0.3% 0.6% 0.3% 0.4% 0.2% 0.4%0.6%

MSS were implanted subcutaneously in the dorsa of adult mice andharvested for immunohistochemistry at 7 and 14 days after implantation.Immunohistochemical analysis identified cellular infiltration in all MSS(FIGS. 3A-3D), with greatest infiltration seen in 1% MS/0.3% H, and 0.6%MS 0.3% H (FIGS. 3C-3D). In addition, CD31 expression was seen in allMSS after 7 and 14 days of implantation (FIGS. 4A-4B), indicative ofinvading endothelial precursors and the formation of neovasculature.

Example 4. MSS Promotes Cellular Infiltration Over 28 Day Implantation

Eighteen mice received four subcutaneous implants (A-D) per mouse asfollows: (A) MSS (1% collagen microspheres in 0.3% collagen bulk), (B)1% bulk collagen hydrogel control, (C) 0.3% collagen hydrogel control,and (D) 7 mm diameter section of INTEGRA Dermal Regeneration Template(Integra LifeSciences, Plainsboro, N.J.). Mice were sacrificed at 7, 14,and 28 days post-implantation (6 mice per time point).

At 7 days after implantation (FIGS. 5A-5D), MSS, 1% collagen control,and INTEGRA scaffolds retained similar size and morphology relative topre-implantation, while 0.3% collagen control was noticeably reduced insize (FIG. 5D). H&E staining of MSS 1 week after implantation revealsinvasion of cells all the way to the center of the scaffold (FIG. 6A).By comparison, there is no invasion of the 1% collagen scaffolds (FIG.6B), except along cracks where the material has split. There was alsominimal invasion into the shrunken 0.3% collagen scaffold (FIG. 6C).Fluorescent staining of the MSS template with CD31 antibodies (toidentify endothelial progenitor cells) and DAPI (to identifyinfiltrating cells) shows that multiple cell types, includingendothelial progenitor cells, are already infiltrating the MSS scaffoldat 7 days (FIG. 7). CD31+ cells were not observed within 1% and 0.3%hydrogel controls (data not shown).

After 14 days (FIGS. 8A-8E), the MSS, 1% collagen control, and INTEGRAscaffolds are still close to pre-implantation size, while 0.3% collagencontrol is dramatically reduced in size (FIG. 8E). The MSS scaffoldshows significant cellular invasion (FIG. 9A), the 1% collagen displaysminimal invasion except along fissures (FIG. 9B), and the 0.3% collagenscaffold shows sparse invasion (FIG. 9C). The INTEGRA scaffold showedless robust invasion than in the MSS scaffold (FIG. 9D); the densestructure of the INTEGRA scaffold led to shearing of the scaffold duringsectioning for H&E staining.

A comparison of cell count per unit scaffold area (FIG. 10) shows thatsignificantly more cells invaded the MSS scaffold at 7 and 14 days(approximately 7 cells and 10 cells per area, respectively) relative to1% hydrogel (approximately 3 and 5 cells per unit area) and 0.3%hydrogel (approximately 3 and 7 cells per unit area).

At 28 days post-implantation (FIGS. 11A-11D), the MSS, 1% collagencontrol, and INTEGRA scaffolds are slightly smaller thanpre-implantation size, while 0.3% collagen control is smaller than at 7or 14 days (FIG. 11D). The MSS scaffold at 28 days shows good cellularinvasion (FIG. 12A), the 1% collagen displays essentially no invasion(FIG. 12B), and the 0.3% collagen scaffold shows invasion despite itssmall size (FIG. 12C). The INTEGRA scaffold showed some invasion as well(FIG. 12D).

Example 5. Scanning Electron Microscopy of Microspheres

Microspheres were prepared as in Example 1 and prepared for scanningelectron microscopy (SEM). As seen in FIG. 13, microspheres can vary insize (between 50-300 μm) and in shape (some are highly spherical, whileothers are irregular in morphology).

What is claimed is:
 1. A method to promote wound healing or tissueregeneration in a subject in need thereof, comprising applying to awound or tissue of the subject a tissue scaffold material comprising ahydrogel and microspheres, said hydrogel comprising a first polymer andsaid microspheres comprising a second polymer, wherein said microspheresare embedded in said hydrogel and have a density of said second polymerthat is at least 25% greater than a density of said first polymer in thehydrogel, wherein said first and second polymers are independentlyselected from the group consisting of collagen, gelatin, elastin,hyaluronate, cellulose, fibrinogen, poly(lactic-co-glycolic acid)(PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA),poly(caprolactone), poly(butylene succinate), poly(trimethylenecarbonate), poly(p-dioxanone), and poly(butylene terephthalate); apolyester amide, a polyurethane, poly[(carboxyphenoxy) propanesebacicacid], poly[bis(hydroxyethyl) terephthalate-ethylorthophosphorylate/terephthaloylchloride], a poly(ortho ester), apoly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester,poly(Beta-hydroxyalkanoate), and a tyrosine derived polycarbonate,wherein said microspheres are embedded in said hydrogel such that theremaining volume in said tissue scaffold material is taken up by saidhydrogel.
 2. The method of claim 1, wherein said first and secondpolymers are collagen.
 3. The method of claim 1, wherein themicrospheres are comprised of 0.2% to 2.0% w/v of said second polymer.4. The method of claim 3, wherein said microspheres are comprised of0.4% to 1.2% w/v of said second polymer.
 5. The method of claim 4,wherein said microspheres are comprised of 0.6% to 1.0% w/v of saidsecond polymer.
 6. The method of claim 1, wherein said microspheres arebetween 50-250 μm in diameter.
 7. The method of claim 1, wherein saidhydrogel is comprised of collagen in an amount of 0.1% to 0.6% w/v. 8.The method of claim 1, wherein said microspheres comprise at least about70% of the volume of tissue scaffold material.
 9. The method of claim 1,wherein said microspheres comprise 0.4 to 1.2% w/v collagen and saidhydrogel comprises 0.2 to 0.6% w/v collagen.
 10. The method of claim 9,wherein said microspheres comprise 0.6-1.0% w/v collagen and saidhydrogel comprises 0.3% w/v collagen.
 11. The method of claim 1, whereinsaid microspheres further comprise bioactive factors.
 12. The method ofclaim 1, wherein said microspheres do not comprise additional bioactivefactors.
 13. The method of claim 1, wherein the tissue scaffold materialis in the form; of a sheet or in a flowable form.
 14. The method ofclaim 13, wherein the tissue scaffold material is in the form of a sheetwith a depth of 0.5-3.0 mm.
 15. The method of claim 14, wherein thetissue scaffold material is in the form of a sheet with a depth of about1.0-2.0 mm.
 16. The method of claim 15, wherein said tissue scaffoldmaterial is applied to an area of said subject with exposed bone,hardware, or necrotic tissue.
 17. The method of claim 1, wherein thetissue scaffold material is comprised in a dressing.
 18. The method ofclaim 1, wherein said microspheres are between 10-500 μm in diameter.